The majority of cancer-related deaths occur after primary therapy has been completed, mostly due to recurrence of the cancer or development of second cancers. Major efforts are underway to develop pharmaceuticals that can prolong the disease-free interval after primary therapy by preventing recurrence of the cancer or the development of new cancers. Only agents that lack significant toxicity are acceptable in this setting. One of the most promising classes of cancer chemoprevention agents designated by the Chemoprevention Working Group to the American Association for Cancer Research (MCR) is retinoids (1). These compounds, which are modeled after the active vitamin A metabolite, retinoic acid (RA), offer promise as cancer chemoprevention agents because of their abilities to regulate growth, differentiation, apoptosis, angiogenesis, metastasis and immune function. Despite limited success of various isomers of RA (All-trans-RA, 13-cis-RA and 9-cis-RA) and a synthetic retinoid in chemoprevention trials (Fenretinide, 4-HPR), structural alterations of the compounds are needed to improve the therapeutic ratio (efficacy/toxicity) before clinical application of a retinoid strategy for chemoprevention (2-5).
The toxicities associated with chronic retinoid treatment affect the skin, mucus membranes, hair, eyes, gastrointestinal system, liver, neuromuscular system, endocrine system, kidneys and bone, and are collectively termed hypervitaminosis A (6). These individual toxicities and teratogenicities have been shown to be induced through activation of nuclear retinoic acid receptors (RARalpha, RARbeta, RARgamma) and retinoid X receptors (RXRalpha, RXRbeta, RXRgamma) that act as transcription factors (7, 8). Early efforts to improve the therapeutic ratio involved constraining the RA double-bonds, by inclusion in an aromatic ring of chemical structures called arotinoids. The first arotinoid evaluated, TTNPB, was 10-fold more potent than RA in biological assays of efficacy, but considerably more toxic (9-12).
Our strategy to reduce the toxicity of arotinoids was to retard metabolic oxidation of the compounds by incorporation of oxygen or sulfur heteroatoms to replace one of the gem-dimethyl groups in the tetrahydronaphthalene ring of TTNPB (FIG. 1A-1B). The resulting compounds, called Heteroarotinoids (Hets), exhibited the similar biological activities to RA (9, 13), but significantly reduced toxicities (12). Thus, inclusion of the heteroatom in the arotinoid structure was shown to greatly improve the therapeutic ratio (efficacy/toxicity) in animal models (9, 12). The clinical application of a Het called Tazarotene (produced by Allergan) for treatment of psoriasis, has confirmed the improved therapeutic ratio predicted for compounds with heteroatoms (14).
Individual structural alterations of Hets greatly affected their selectivities for individual RAR and RXRs (FIG. 1) (12, 15-17). A Het that activated RXRs only (OHet72) was found to be sufficient to inhibit establishment head and neck xenograft tumors, while a retinoid that activated both RARs and RXRs (SHet50) exerted greater growth inhibitory activity (17). The importance of RARgamma activation in skin cancer was demonstrated by comparisons of Hets, which differed by single structural alterations that regulated their abilities to activate the RARg receptor. A Het that activates all six nuclear receptors (NHet90) induced significantly greater growth inhibition of vulvar carcinoma cell lines in comparison to structurally related compounds, that activate all retinoid receptors except RARgamma (NHet17 and NHet86) (16). Interestingly, Hets containing three-atom urea or thiourea linkers, which increased the flexibility of their conformations, regulated growth and differentiation similar to RA, but did not activate the RARs and RXRs (18). These flexible Hets (Flex-Hets) exhibited significantly greater growth inhibition activity against epithelial ovarian cancer and borderline-cancer cells than benign epithelial ovarian cells and normal endometrial cells (19). The most potent Flex-Het, SHetA2, was characterized for the mechanism of this strong growth inhibition in head and neck cancer cell lines, and was found to induce apoptosis through G2 cell cycle arrest, alterations in mitochondrial membrane permeability, release of cytochrome c from the mitochondria, generation of reactive oxygen species (ROS), and activation of caspase 3 (19, 20). Generation of ROS was also demonstrated in SHetA2-treated ovarian cancer cell lines (19).
While natural RA isomers and classical retinoids are weak apoptosis inducers, some retinoids, 4-HPR, CD437/AHPN and MS3350-1, which are selective for RARgamma activation, exhibit potent apoptosis-inducing activity similar to Flex-Hets (21). 4-HPR also weakly activates RARbeta, and activation of multiple retinoid receptors by 4-HPR is involved in the mechanism of growth inhibition in leukemia cells (22, 23). The additional non-retinoid activities possessed by these compounds have led to their classification as retinoid-related molecules (RRMs). While the ability of these compounds to induce apoptosis is only partially independent of the retinoid receptors, Flex-Hets are unique in that they induce apoptosis completely independent of RAR and RXR activation (20, 24, 25). Several clinical trials of 4-HPR demonstrated limited cancer chemoprevention activity at low doses, and tolerable toxicity at higher doses sufficient to induce apoptosis (3, 5, 26).
Identification of therapies for inhibiting angiogenesis is an important area of research. A multitude of experiments have demonstrated that the development of blood vessels is required to support tumor growth and metastasis. Studies of human tumor specimens found that the number of vessels within tumors correlates with disease stage and patient prognosis. The levels of circulating angiogenic cytokines also correlate with prognosis in cancer patients. The migration of endothelial cells to form blood vessels within tumors is a complex process involving cancer cells, growth factors, fibroblasts, extracellular matrix turnover. Physical contact between endothelial cells and fibroblasts is required for the differentiation of endothelial cells into tubes with branches.
Strategies for therapeutic angiosuppression generally involve either interference with the activators of angiogenesis or amplification of the endogenous suppressors. The classes of angiogenesis antagonists in current clinical trials include inhibitors of proteases, endothelial cell migration and proliferation, angiogenic growth factors, matrix proteins on the endothelial cell surface, such as integrins, copper antagonists, and other inhibitors with unique mechanisms.
For example angiogenic inhibitors in current clinical trials include (1) protease inhibitors such as marimastat, BAY 12-9566, Af 3340, and Neovastat, (2) inhibitors of endothelial cell migration and proliferation such as TNP-470, squalamine, combretastatins, endostatin, angiostatin, penicillamine, (3) antagonists of angiogenic growth factors such as anti-VEGF antibody, thalidomide, sugen-5416, antiangiogenic ribozyme, SU 6668, interpheron-alpha and suramin, (4) inhibitors of endothelial-specific Integrin/Survival signaling such as Integrin antagonists (e.g. Vitaxin), (5) copper antagonists/chelators such as penicillamine, tetrathiomolybdate and captopril, and (6) angiogenic inhibitors with distinct mechanisms such as ABT-627, CM101, Interleukin-12, IM862 and PNU145156E.
Other areas of research involve therapies for treating lysosomal storage diseases and polycystic kidney disease.
Several approaches to develop experimental model systems for the study of endometrium have been attempted. The earliest studies of endometrium were performed using animal models such as the Rhesus monkeys. In human studies, organ cultures cut from hysterectomy specimens have been used as a model system, but were limited by variability between specimens and inability to cycle the endometrium in vitro. Cultured endometrial cells collected from hysterectomy specimens, peritoneal fluid or curretage of the endometrium have also been utilized after separating the stromal cells from the epithelial glands. More recently, cells collected from menstrual secretions have been successfully grown in tissue culture. Although these studies have been successful at culturing normal endometrial cells in monolayers, the systems used are highly artificial and do not mimic the in vivo architecture or intracellular communication of the human endometrium. Three dimensional organotypic cultures of endometrial cells isolated from surgical specimens and grown in collagen I or basement membrane material (Matrigel) have been used in attempts to develop experimental systems for the study of human endometrium. These cultures demonstrated characteristics of endometrial architecture but were limited by only occasional formation of gland-like structures and considerable shrinkage of the collagen gels over prolonged treatment times.
In addition to steroid hormones, retinoic acid, the natural metabolite of vitamin A, is involved in the maintenance and regulation of differentiation in the cycling endometrium and the decidualization of fibroblasts. Throughout the menstrual cycle, the intracellular levels of retinoic acid and expression of cellular retinoid binding proteins fluctuate, while nuclear retinoic acid receptors remain at similar levels. Epidemiological studies have found that high dietary intake of vitamin A and carotenoids have been associated with a decreased risk for endometrial and ovarian cancer. Retinoic acid was shown to delay tumor induction in hamster cheek pouch epithelium exposed to 25 μg DMBA. The papillary epidermoid carcinomas that developed were less invasive and less keratinized in the retinoic acid treated animals than in the animals treated with DMBA alone.
Autosomal dominant polycystic kidney disease is one of the most common inherited disorders in humans, occurring in 1 in 500 to 1 in 1000 individuals. The cost of this disease is enormous as it is the third leading cause of end-stage renal disease. Costs are further escalated by extra-renal complications such as hypertension, cystic liver disease and intracranial aneurysms. Therefore, any therapy that can slow the progression of this disease would be of great benefit.